Retromer contributes to the invasive properties of breast cancer cells. (A) The KD efficiency of the respective genes was confirmed by Western blot or quantitative PCR (qPCR; n = 3). Values in the graph represent means ± SEM. Two-tailed Student’s t test, ** P < 0.01. (B) Retromer, MT1-MMP, or STX8 were depleted via siRNA and cells were seeded on Alexa Fluor 568–labeled gelatin-coated coverslips for 12 h. Degradation activity was quantified as degradation index, described in the Materials and methods (N = 4, n = 300; scale bar = 10 µm). Arrowheads represent degradation spots. One-way ANOVA, *** P < 0.001. The graph represents means ± SEM. (C) Cells depleted for indicated molecules were seeded on Matrigel-coated cell inserts and allowed to invade for 20 h. Invasive cells were counted and the percentage of invaded cells were plotted (N = 3, scale bar = 30 µm). One-way ANOVA, *** P < 0.001. The graph represents means ± SEM. (D and E) MDA-MB-231 cells treated with indicated siRNA were immunostained for invadopodia markers cortactin (D) or Tks5 (E) and stained with phalloidin to label actin. Images were analyzed on the confocal microscope and the percentage of cells forming invadopodia was quantified and plotted (N = 3, n = 400; scale bar = 10 µm, inset = 4 µm). The inset represents the actin-cortactin– or Tks5-actin–rich invadopodia. Invadopodia (Tks5/actin dots) per cell were counted (N = 3, n = 50). The graph represents means ± SEM. One-way ANOVA, ** P < 0.01, *** P < 0.001. N, number of experimental repeats.